Traditionally, adventitious agent contamination, viability and infectivity has been investigated using in vitro culture methods, cell culture-based infectivity assays, electron microscopy measurements and more recently DNA-specific polymerase chain reaction (“PCR”) methods. Cell culture-based infectivity assays are expensive, time consuming and labor intensive. PCR assays on the other hand are fast, accurate and specific, but currently do not distinguish between “viable” and non-viable adventitious agent contamination. European Patent Publication No. 0 464 010 A describes a polymerase chain reaction protocol for detection of bovine viral diarrhea virus in biological specimens. Likewise, World Patent Publication No. WO 96/36735 describes a rapid method for detection of the presence of a specific mycoplasma in a nucleic acid sample. However, neither of the disclosed methods is able to discriminate between infectious and non-infectious viruses or viable or non-viable mycoplasma, respectively. World Patent Publication No. WO 99/23203 discloses a method for detecting the presence of a virus in a sample. However, as set forth in that publication, the method is restricted to viral detection and requires the use of an immortalized cell line. Moreover, as is the case with the assays described in EP 0 464 010 A and WO 96/36735, the method described in WO 99/23203 is incapable of discriminating between of infectious and non-infectious viruses.
Detection of viability, infectivity and contamination is important in process validation and control of therapeutics such as vaccines and biotechnology products. Testing of cell lines, raw materials and manufacturing processes can greatly benefit from development of fast and reliable means of detecting viable and infectious adventitious agents.